1. Field of the Invention to previously isolated hydantoinases and their use in whole-cell catalysts to manufacture amino acids.
2. Description of Related Art
Hydantoin hydrolyzing enzymes, which will be referred here as xe2x80x98hydantoinasesxe2x80x99, comprise a diverse class of enzymes having a wide range of specificities and biological functions. Some hydantoinases for example play an essential role in the reductive pathway of pyrimidine degradation (dihydropyrimidinases, EC 3.5.2.2) whereas others catalyze reactions in the purine degradation pathway (allantoinases, EC 3.5.2.5). Despite their functional diversity hydantoinases show significant sequence similarities and belong to a superfamily of amidohydrolases related to ureases as described by Holm, L., and Sander, C. (1997) An evolutionary treasure: Unification of a broad set of amidohydrolases related to ureases, Proteins 28:72-82; The alignment of sequences from the different hydantoinases was used to identify conserved residues that are important for catalytic function as described by May, O., Habenicht, A., Mattes, R., Syldatk, C. and Siemann, M. (1998) Molecular Evolution of Hydantoinases, Biol Chem. 379:743-747; and Kim, G. J. and Kim, H. S. (1998) Identification of the structural similarity in the functionally related amidohydrolases acting on the cyclic amide ring. Biochem. J. 330:295-302. Despite this knowledge that allows to identify equivalent amino acid residues of the different hydantoinases, knowledge about the function of other amino acid residues is limited. So far, no X-ray structure of hydantoinases was reported.
An important property of hydantoinases is their enantioselectivity which makes them valuable for the production of optically pure D- or L-amino acids. A detailed background of hydantoinases is provided in the published doctoral thesis of Oliver May entitled xe2x80x9cThe Hydantoinase from Arthrobacter aurescens DSM 3745 and its Relation to other Hydantoinasesxe2x80x9d (Institut fuer Bioverfahrenstechnik, Lehrstuhl Physiologische Mikrobiologie, Universitaet Stuttgart-1998).
In view of the importance of hydantoinases to the production of optically pure amino acids, there has been a concentrated effort to develop modified enzymes which have improved properties with respect to amino acid production. As a result of this effort, a number of microorganisms have been isolated and identified which produce hydantoinases with desirable enzymatic properties. U.S. Pat. No. 5,516,660 discloses microoganisms identified as DSM7329 and DSM 7330 which produce hydantoinases that are capable of producing L-alpha-amino acids from D-, L- and/or D,L-5-monosubstituted hydantoins. In U.S. Pat. No. 5,714,355, a mutant of the DSM 7330 microorganism is disclosed which has greater enzymatic activity than the parent organism by a factor of up to 2.7. The mutant (DSM 9771) was obtained by cultivating the parent DSM 7330 organism under selective pressure using L-carbamoylmethionine (L-CAM) as the sole source of nitrogen. Although the hydantoinases produced by the above-mentioned microoganisms are well-suited for at least some of their intended purposes, there still is a continuing need to develop new enzymes which exhibit even more desirable hydantoinase activity. In particular, there is a need to improve enantioselectivity as well as catalytic activity.
In accordance with the present invention, modified hydantoinases are provided which have enhanced enzymatic properties (better whole-cell catalysts) with respect to the hydantoinase produced by the microorganism DSM 9771 which is identified in U.S. Pat. No. 5,714,355. The DSM 9771 hydantoinase has an amino acid sequence which includes numbered positions ranging sequentially from 1 to 458 (SEQ. ID. NO. 2).
It was discovered that substitution of amino acids at one or more specific amino acid positions within the DSM 9771 enzyme resulted in the formation of enzymes having enhanced properties with respect to activity and enantioselectivity. The specific amino acid position numbers at which substitutions are made to achieve the modified hydantoinase enzymes in accordance with the present invention are positions Nos. 95, 154, 180, 251 and 295. As a further feature of the invention, specific amino acid substitutions at the various positions are identified to provide specific types of modified hydantoinases. The specific amino acid substitutions include 195F, I95L, V154A, V180A, Q251R and V295A. One or more of these specific substitutions were found to enhance the enzymatic activity and change the enantioselectivity of the xe2x80x9cwild typexe2x80x9d DSM 9771 hydantoinase. These changed enzyme properties were found to contribute to a significantly improved hydantoinase process by reducing the accumulation of the wrong enantiomer of the N-carbamoyl-amino acid.
Six specific modified hydantoinases are disclosed which have one or more of the above amino acid substitutions. The amino acid sequences for these modified hydantoinases are set forth in SEQ. ID. NOS. 4, 6, 8, 10, 12 and 14. These modified hydantoinases are also identified throughout the specification as 1CG7, 11DH7, 1BF7, 19AG11, 22CG2 and Q2H4, respectively.
It was further discovered that hydantoinases evolved for activity and/or enantioselectivity can dramatically improve the production of amino acids (i.e., L-methionine) using a whole cell catalyst comprising an evolved hydantoinase in addition to at least a carbamoylase.
The above discussed and many other features and attendant advantages of the present invention will become better understood by reference to the following detailed description when taken in conjunction with the accompanying drawings.